1. Field of the Invention
This invention relates to a container for intravaginal fertilization and culture of mammalian, and in particular human, oocytes and an embryo transfer method employing such a container.
2. Description of Prior Art
Conventional in vitro fertilization (IVF) techniques are notoriously complex and have tended to become even more so since intracytoplasmic sperm injection (ICSI) was added to the panoply of assisted reproductive technologies.
Typically, IVF involves aerobic and sterile culturing of embryos in Petri dishes at 37.degree. C. in a 5% CO.sub.2 enriched atmosphere. It requires cumbersome and expensive equipment such as a CO.sub.2 incubator controlled at 37.degree. C. 24-hours a day, during the two or three days required for fertilization and culture.
IVF also involves the use of many pieces of disposable equipment and media for the numerous steps carried out over the 48 or 72 hours of treatment of gametes and embryos in the CO.sub.2 enriched incubator. Many include delicate manipulations requiring considerable skill and dexterity of a laboratory biologist. One of these consists in denuding the embryo of cumulus cells prior to implantation.
One of the co-inventors, Claude Ranoux, developed a new procedure known as intravaginal culture (IVC) which comprises maturation of gametes, fertilization and embryo development in a sealed container filled with a suitable culture medium and placed in the vaginal cavity which serves as an incubator. This procedure and the fertilization and culture container are disclosed in U.S. Pat. No. 4,902,286 issued Feb. 20, 1990 and U.S. Pat. No. 5,135,865 issued Aug. 4, 1992.
All IVC procedures for experimental and clinical purposes have to date been performed using a polypropylene cryotube, designated by reference numeral 100 as shown in FIG. 2, manufactured by Nunc, of Kamptrup, Denmark and hermetically closed and sealed in a polyethylene cryoflex envelope 101 also manufactured by Nunc. The cryotubes and associated cryoflex envelopes were designed for cryopreservation of tissue specimens. The Nunc cryotube and cryoflex envelope have considerable drawbacks when used to perform IVC.
Among these drawbacks is the fact that the tube has to be filled virtually to the free edge or brim of the (externally threaded) tube neck to avoid the formation of air bubbles which could otherwise interfere with fertilization. Also in practice, sperm must be introduced into the tube before the oocytes because the turbulence produced during sperm injection into the culture medium might otherwise carry one or more oocytes to the surface of the medium and cause them to spill out over the brim. The cumulus of the oocytes have some occluded air bubbles which may cause them to float to the surface of the culture medium only to be lost when the cap 103 is screwed on to the neck of the tube.
Likewise, when the threaded cap is screwed on to the neck, there is a risk of culture medium overflow, entraining one or more oocytes out of the tube.
The cryoflex envelope 101 has to be sealed around the tube to protect the tube from contamination during vaginal residence. In practice, this requires heating a crimping tool or pliers and softening and crimping each of the ends of the tube to form serrated welds or seals 104. The free edges of the welds in turn must then be clipped and/or shaped with a cutter to eliminate corners, but this does not avoid all discomfort or even injury in the course of lodgment in the posterior fornix as the free transverse edges of the seals remain both rough and sharp. Also, a single size tube cannot be used for all individuals owing to anatomical incompatibility.
Many of these drawbacks of the Nunc cryotube are obviated by the container disclosed in Ranoux U.S. Pat. Nos. 4,902,286 and 5,135,865. The container body comprises a membrane or valve which prevents the loss of oocytes during their introduction or the introduction of sperm. Moreover, the membrane or valve limits the area of contact between the culture medium and the surroundings and tends to maintain the pH of the culture medium stable. These patents also disclose a flexible rubberized ring from which is appended a rubber pouch for accommodating the container. The diameter of the ring has to be determined individually, like a diaphragm, as a function of the size of the cervix. The tube is intended to be held in the posterior fornix of the vagina but may drop out and then held dangling from ring and out of contact with the vaginal walls.
The development of IVC has been hindered by the lack of a container the use of which is simple and safe, and which does not involve the high level of laboratory skill that have been required to date and which can avoid transfer of the embryos from the container to a Petri dish for microscopic inspection before their ultimate transfer to the uterine cavity.
To be sure, Ranoux U.S. Pat. No. 5,084,004 discloses an intra-uterine fertilization container with an exit area for access to the uterine cavity and transfer of one or more fertilized oocytes from the container to the cavity. Such a device is unsuitable for use in the vagina owing to the lack of sterile conditions liable to contaminate the embryos. Moreover, the preferred embodiments involve the use of a special holding device which is introduced into the cervix and a second section located in the vagina external to and abutting the cervix. A piston is actionable from inside the vagina to expel the contents of the container into the uterine cavity. This device is complicated by the incorporated embryo ejection means and does not admit readily of microscopic examination of the embryos prior to ejection. Moreover, it is not possible to simply and precisely locate embryos in the container for examination.